alpha-L-Rhamnosidases with desirable activity and thermostability profiles could be used for the biocatalytic production of the flavonoid glucoside isoquercetin from natural rutin for functional food. Herein, to improve the catalytic activity of GH78 alpha-L-rhamnosidase BtRha78A from Bacteroides thetaiotaomicron VPI-5482, a list of residues located at the conserved general acid motif were selected for targeted mutagenesis by the sequence alignment of BtRha78A with homologous alpha-L-rhamnosidases. Ala-scanning mutagenesis and site-directed mutagenesis based on sequence alignment were performed, and the relative activity on rutin was evaluated. Furthermore, the reaction time curves and enzyme kinetics of better mutants were determined. The results indicate that the conversion rates of mutants V338A, V338I, S340A, and G341A were increased by 21.3%, 20.1%, 13.2%, and 1.6%, respectively, compared with the wild type when using whole-cell biotransformation. Moreover, the catalytic efficiency k(cat)/K-M value of mutant V338A was 1.3-fold higher than that of the wild type. The best mutant, V338A, was employed for the enzymatic preparation of isoquercetin via the biotransformation of rutin at a concentration of 2 mM, and 1.80 g of isoquercetin was obtained. The identification of the best mutant V338A lays the foundation for the efficient preparation of isoquercetin via the biotransformation of rutin, which in turn provides theoretical guidance for its large-scale production.
